The present invention relates to methods of preserving functionality of an organ, preserving fertility of a patient undergoing a treatment expected to cause sterility and assuring a supply of viable gametes for future use and, more particularly, to a methods which rely upon removal of an organ, cryopreservation of the removed organ and reintroduction of the cryopreserved organ into a recipient in such a way that the vasculature of the recipient supplies blood to the introduced organ or a portion thereof.
Undoubtedly, successful cryopreservation of solid organs of clinical interest would have a significant impact on the field of organ transplantation. However, despite several decades of research freezing and thawing of solid organs remains largely impractical (Dietzman et al., 1973; Guttman et al., 1977; Pegg et al., 1987; Karow, 1981; Jacobsen et al., 1982).
Attempts to understand the mechanism by which cold-blooded animals survive freezing in nature (Storey and Storey, 1988; Storey, 1990) have led to successful, short-term cryopreservation of hearts and livers by freezing at high subzero temperatures (−3.4° C. to −4° C.), (Wang et al., 1992; Banker et al., 1991, Rubinsky et al., 1994, Soltys et al., 2001).
Cryopreservation at lower temperatures, which is necessary for long-term storage, was attempted in dog intestines, but vascular injury was observed after thawing (Karlasson et al., 1996). Livers have regained partial function after freezing to −60° C. (Mazur, 1977), dog spleens (Fuller, 1987) and ureters (Pegg, 1987) have survived deep freezing and transplantation, and kidneys have suvived freezing to −80° C.
(Fahy et al.,1984) proposed vitrification (ice free cryopreservation) as an alternate strategy for for organ cryopreservation (Kheirabadi et al., 2000) . Vitrification produced a high survival rate in cryopreserved small organisms such as Drosophila embryos at −196° C. (Steponkus et al., 1990; Mazur et al., 1992).
Thus, successful cryopreservation of organs apparently requires a specific optimal cooling rate, because damage may occur if organs are frozen either too rapidly or too slowly (Karisson and Toner., 1996; Mazur, 1977). Cooling too slowly (<1° C./min) causes extracellular crystallization which may physically disrupt the vital tissue architecture (Fuller, 1987; Pegg, 1987), whereas at higher cooling rates intracellular crystallization will cause irreversible damage.
Further, cryopreservation of large-volume samples, such as tissues or organs, introduces heat transfer problems. In macroscopic samples there is a large thermal gradient from the surface of the sample to the interior. For example, it was shown that cells survived equally when frozen as isolated cells or in a monolayer, only when the applied cooling rate was less than 0.5° C./min. Moreover, the survival of cells in monolyer was higher than isolated cells using the determined optimal cooling rate of 0.2° C./min. This indicates that the attached cells were more tolerant of slow cooling injury (Armitage et al., 1996). The need for a slow cooling rate is further illustrated by the cooling rate at which a Wood frog survives freezing (less than 0.1° C.,/minute; Schmid, 1982).
Typically, prior art freezing techniques produce temperature gradients within the freezing chamber which make it difficult to achieve an optimal cooling rate (Koebe et al., 1993). In addition, cooling rates slower than 0.1° C./minutes are hard to control in a programmable freezing apparatus since the accuracy of the temperature measurement is within that range (OMEGA ENGINEERING, INC). U.S. Pat. No. 5,873,254 to Arav teaches a device capable of producing a uniform cooling rate of 0.1° C./min throughout a bilogical sample. However, the earlier teachings of Arav do not disclose methods for cryopreservation of whole organs and subsequent introduction of those whole organs into a recipient subject. Specifically, the earlier teachings of Arad do not include methods for thawing a whole organ without impairing functionality thereof and surgical techniques for anastomic transplantation.
It is well established that treatment of a malignant disease by radiotherapy or chemotherapy can have dramatic and irreversible effect, on fertility, especxially in female patients (Byrne et al., 1987; Ataya, 1989; Familiari, 1993).
Thus, there is a recognized need, but no established method, for cryopreservation of a gonadal organ for subsequent reimplantation for patients at risk for premature sterility as a result of planned cancer treatment. While oocyte cryopreservation theoretically offers a means of preserving fertility for these patients, severe practical problems for oocyte cryopreservation remain unsolved (Arav et al., 1996; Zeron et al., 1999; Zenzes et al., 2001). Similarly, while cryopreservation of semen for male patients is available, it is not an optimum method of assuring male sterility. Cryopreservation of oocytes and/or semen typically require laboratory intervention in the form of IVF procedures. This prospect is daunting to many patients for a variety of reasons including emotional and religous reasons. Thus, storage and later use of of a gonadal organ for conception by conventional methods offers significant advantages over previously available alternatives.
A high proportion of viable follicles have been found to survive in human ovarian tissue after freeze-thawing (Hovatta et al., 1996; Newton et al.,1996; Oktay et al., 1997), and this has aroused interest in the procedure as a potential means of preserving the fecundity of patients at risk of premature ovarian failure (Donnez et al., 1998; Newton et al., 1998). However, freezing and thawing of the whole ovary was not reported.
Gosden and colleagues (1994) have achieved limited success in sheep using ovarian cortex freezing leading to speculation that this technique may be applicable to humans. The sheep ovary is similar to the human ovary in that it has a dense fibrous stroma and relatively high density of primordial follicles in the ovarian cortex.
Autotransplantation of frozen-banked and fresh ovarian cortex cryopreservation ovarian cortex, have resulted in two pregnancies (Gosden et al., 1994). Baird et al. (1999) performed frozen tissue autotransplantation in eight sheep, which were monitored for up to 22 months. All the animals resumed cyclicity and showed hormone production, however, it was established that there was a drastic reduction in the total follicular number, and the resumption of cyclical ovarian function was temporary. Thus, despite recent advances in this area, transplantation of a functional portion of an ovary for purposes of restoring full ovarian function remains unreliable. Therefore, freezing and grafting of human ovarian tissue is not considered clinical option (Kim et al., 2001).
Clinical acceptability of ovarian tissue transplantation will require higher numbers of follicles survive and retain the capability to develop and ovulate. Recently it has been demonstrated that during freezing of cortical ovarian slices granulosa cells are more damaged than oocytes (Siebzehnrubl et al., 2000).
Further, currently accepted experimentyal practice idictates use of ovarian slices resulting in schemic damage as a rersult of non-vascular transplantation. Still further, the need for an IVF procedure in addition to preparing tissue for cryopreservation is time consuming and increases costs of the procedure. Finally, in those cases where ovarian function has been restored, long post=implantation delays are observed (Radford et al., 2001).
Transplantation of whole ovary including vascular has been recognozied as a theoretical method of solving the problems described above. While Ovary transplantation has been known for decades, (Nobel Lecture, Dec. 11, 1912:” Suture of blood-vessels and transplantation of organs”), autotransplantation has been considered impractical because of the absence of long term organ cryopreservation.
There is thus a widely recognized need for, and it would be highly advantageous to have, methods of preserving functionality of an organ, methods of preserving fertility of a patient undergoing a treatment expected to cause sterility and methods of assuring a supply of viable gametes for future use devoid of the above limitation.